KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres

Bosco, Nazario; Goldberg, Aleah; Zhao, Xin; Mays, Joseph C.; Pan, Chuyue; Johnson, Adam F.; Bianchi, Joy J.; Toscani, Cecilia; Tommaso, Elena Di; Katsnelson, Lizabeth; Annuar, Dania; Mei, Sally; Faitelson, Roni E.; Pesselev, Ilan Y.; Mohamed, Kareem S.; Mermerian, Angela; Camacho-Hernandez, Elaine M; Gionco, Courtney A.; Manikas, Julie; Tseng, Yi-Shuan; Sun, Zhengxi; Fani, Somayeh; Keegan, Sarah; Lippman, Scott M.; Fenyö, David; Giunta, Simona; Santaguida, Stefano; Davoli, Teresa

doi:10.1016/j.cell.2023.03.029

Cited by 30 publications

(36 citation statements)

References 84 publications

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“…To our surprise, sgChr20-3 induced 9.2% losses and 18.5% gains in chr2 (p < 0.001), which we may not have observed if we had instead evaluated the gRNA using a chromosome-targeted method such as fluorescence in situ hybridization rather than a genome-wide method. The sgChr20-3 sequence matches 18 out of 20 base pairs for sites on the chr2 centromere 12 , suggesting an explanation of the off-target effect. These data indicate that our panel can be used to efficiently screen CRISPR genome engineering methods like KayroCreate and successfully use gRNA detection to multiplex samples.…”

Section: Resultsmentioning

confidence: 87%

“…To demonstrate the combined copy number detection and multiplexing capabilities of our system, we tested it on samples treated with KaryoCreate, a method we recently developed to induce chromosome-specific aneuploidy in cultured cells 12 . KaryoCreate uses CRISPR gRNAs to target a mutant KNL1-dCas9 fusion protein to the centromere of a specific chromosome, causing missegregation in ∼20% of cells.…”

Section: Resultsmentioning

confidence: 99%

“…These data can be used to reassemble the evolution of the tumor's cells, which can provide insights into how aneuploidy and chromosomal instability may drive tumorigenesis or inform treatments for therapeutic resistance 6,9 . Methods for modeling aneuploidies of specific chromosomes in cell culture have also emerged as powerful tools for studying the effects of aneuploidy in cancer [12][13][14] . These methods have benefitted from scDNA-seq, as sequencing individual cells enables the evaluation of the specificity and accuracy of the engineered karyotypes 13,14 .…”

Section: Introductionmentioning

confidence: 99%

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KaryoTap Enables Aneuploidy Detection in Thousands of Single Human Cells

Mays,

Mei,

Bosco

et al. 2023

Preprint

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Investigating chromosomal instability and aneuploidy within tumors is essential for understanding their contribution to tumorigenesis and developing effective diagnostic and therapeutic strategies. Single-cell DNA sequencing (scDNA-seq) technologies have enabled such analyses, revealing aneuploidies specific to individual cells within the same tumor. However, scaling the throughput of these methods to identify aneuploidies occurring at low frequencies while maintaining high sensitivity has been difficult. To overcome this, we developed KaryoTap, a method combining custom targeted panels for the Tapestri scDNA-seq platform with a Gaussian mixture model analysis framework to enable detection of chromosome- and chromosome arm-scale aneuploidy in all human chromosomes across thousands of single cells simultaneously. This system will prove a powerful and flexible resource for the study of aneuploidy and chromosomal instability in tumors and normal tissues.

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

KaryoTap Enables Aneuploidy Detection in Thousands of Single Human Cells

Mays,

Mei,

Bosco

et al. 2023

Preprint

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“…The fact that we observed GFPpositive bridges during anaphase in ∼ 30% of the RPE1-dCas9-Kin14VIb cells transduced with the Chr9-cen sgRNA, but without Kin14VIb binding to the repeat, underscores this notion. We consider it likely that the mere binding of dCas9 to the recently published centromere-specific repeats induces similar replication issues (Bosco et al, 2023). Importantly, the inducible FKBP12-FRB dimerization modality and the presence of a NES in Kin14VIb enabled a direct assessment of the consequence of Kin14VIb motor binding to a chromosome of interest during mitosis.…”

Section: Opportunities and Limitations Of The Approachmentioning

confidence: 85%

A kinesin‐based approach for inducing chromosome‐specific mis‐segregation in human cells

et al. 2023

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Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type‐specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis‐segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease‐dead Cas9 to link a microtubule minus‐end‐directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome‐specific endogenous repeats, respectively. By live‐ and fixed‐cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb‐mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore‐attached microtubules. This tug‐of‐war resulted in chromosome‐specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single‐cell whole‐genome sequencing, we established that kinesin‐induced targeted mis‐segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin‐based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue‐specific aneuploidy patterns evolve.

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“…KaryoCreate (karyotype CRISPR‐engineered aneuploidy technology), a method that allows the production of chromosome‐specific aneuploidies in human cells, was recently developed (Bosco et al ., 2023). Its foundation is the use of dCas9 coupled to mutant KNL1 to target chromosome‐specific CENPA‐binding satellite repeats.…”

Section: A Bright Future For Plant Chromosome Engineeringmentioning

confidence: 99%

Plant chromosome engineering – past, present and future

Puchta,

Houben

2023

New Phytologist

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SummarySpontaneous chromosomal rearrangements (CRs) play an essential role in speciation, genome evolution and crop domestication. To be able to use the potential of CRs for breeding, plant chromosome engineering was initiated by fragmenting chromosomes by X‐ray irradiation. With the rise of the CRISPR/Cas system, it became possible to induce double‐strand breaks (DSBs) in a highly efficient manner at will at any chromosomal position. This has enabled a completely new level of predesigned chromosome engineering. The genetic linkage between specific genes can be broken by inducing chromosomal translocations. Natural inversions, which suppress genetic exchange, can be reverted for breeding. In addition, various approaches for constructing minichromosomes by downsizing regular standard A or supernumerary B chromosomes, which could serve as future vectors in plant biotechnology, have been developed. Recently, a functional synthetic centromere could be constructed. Also, different ways of genome haploidization have been set up, some based on centromere manipulations. In the future, we expect to see even more complex rearrangements, which can be combined with previously developed engineering technologies such as recombinases. Chromosome engineering might help to redefine genetic linkage groups, change the number of chromosomes, stack beneficial genes on mini cargo chromosomes, or set up genetic isolation to avoid outcrossing.

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KaryoCreate: A CRISPR-based technology to study chromosome-specific aneuploidy by targeting human centromeres

Cited by 30 publications

References 84 publications

KaryoTap Enables Aneuploidy Detection in Thousands of Single Human Cells

KaryoTap Enables Aneuploidy Detection in Thousands of Single Human Cells

A kinesin‐based approach for inducing chromosome‐specific mis‐segregation in human cells

Plant chromosome engineering – past, present and future

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